Our overall objective is to determine how chromosome structure and chromatin remodeling enzymes influence genome stability. In particular, we are interested in how these factors regulate the repair of DNA double strand breaks (DSBs) by homologous recombination (HR) and how they control the progression and stability of replication forks. Defects in either of these pathways directly impact cell survival and maintenance of genome integrity, leading to mutations, gene translocations, gross chromosomal rearrangements, or cellular lethality. During the past budget period, biochemical assays were developed to dissect activities of the Sgs1/BLM and Exo1 DNA processing enzymes on chromatin substrates, and we also identified pathways that control recruitment of chromatin regulators to DSBs. In addition, the conserved INO80-C chromatin remodeling enzyme was shown to regulate the stability of stalled replisomes and to exhibit histone dimer exchange activity. Our general strategy is to continue to exploit a powerful combination of biochemical and molecular genetic approaches to dissect the dynamics of chromatin structure during the repair of DSBs and during the replication process, using budding yeast as the experimental system. Experiments described in this proposal address four aims. The first aim investigates the role of chromatin in the early steps of HR. This aim exploits both ensemble biochemical studies as well as single molecule, fluorescent DNA curtains to monitor how the Sgs1/Dna2 machinery disrupts nucleosome structures. Here we also develop yeast strains that allow for conditional depletion of multiple chromatin regulators, and such strains will be used in live cell imaging studies to investigate roles for chromatin regulators in he nuclear mobility of DSBs and the completion of the homology search step of HR. Aim 2 dissects the function of the Ies6/Arp5 module of the INO80-C remodeling enzyme, employing a combination of EM, chemical cross-linking coupled to mass spectrometry, and in vitro chromatin remodeling assays to dissect its role in the dimer exchange reaction. Studies described in Aim 3 investigate recruitment and function of INO80-C at replication origins and stalled forks. A combination of conditional depletion of preRC components, nucleosome mapping, ChIP, and 2D gel analyses are used. Aim 4 describes a combination of analytical ultracentrifugation, AFM, and EM studies to analyze how the Sir2/Sir4 complex interacts with nucleosomal arrays, and we probe the structure and solution dynamics of a reconstituted Sir2/Sir3/Sir4 heterochromatin fiber.